Hox genes encode homeodomain-containing transcription factors that are expressed and function along the body axis in an AP-restricted manner corresponding to their position within the cluster. This pattern of global Hox expression is termed colinearity and this feature of Hox expression has been retained among vertebrate embryos. In addition to the colinearity along the main body axis, a nested, colinear expression pattern is also observed for the HoxA and HoxD paralogous group 9 through 13 genes in the early developing vertebrate limb bud. Mutations in these genes cause specific morphological perturbations along the proximodistal (PD) axis during limb development. The preponderance of evidence supports a model in which only the AbdB-related Hox genes (Hox9-13) from the HoxA and HoxD complex are required in the developing forelimb. No loss-of-function mutants in HoxB or HoxC group genes have ever been demonstrated to have forelimb defects. Similarly, no limb phenotypes have been reported with loss- of-function of non-AbdB-related genes (Hox1-8) from any of the four HoxA-D complex genes. Recent work has additionally shown that Hox10 through Hox13 genes from the HoxA and D complex, together, control early limb bud outgrowth and the initiation of Shh expression, suggesting that the same Hox compliment of genes that control later limb proximodistal patterning events, together, control early outgrowth and anterior-posterior patterning events. It has been clearly shown that colinear expression in the limb relies on regulatory elements both within and outside the Hox cluster, but the factors that control the initiation of the Shh/Hoxa10-13;Hoxd10-13 regulatory loop are unknown. We have recently generated paralogous mutant mice in the Hox5 (Hoxa5 -/-;Hoxb5 -/-;Hoxc5 - /-), and Hox9 (Hoxa9 -/-;Hoxb9 -/-;Hoxc9 -/-;Hoxd9 -/-) group genes. Loss of Hox5 function (non- AbdB-related Hox genes) results in anterior patterning defects of the forelimb skeleton that closely resemble Holt-Oram Syndrome patients, and preliminary evidence suggests Hox5 genes are involved in the Tbx5 regulatory pathway in forelimb development. Also unexpectedly, loss of all four Hox9 paralogous genes results in profound PD outgrowth and AP patterning defects that affect the entire forelimb skeleton. Preliminary results demonstrate that Shh and AbdB-related HoxA/D 10-13 genes are not initiated in Hox9 limb buds, suggesting Hox9 is a node, critical for establishment of the Hox10- 13/Shh loop in forelimb development. The emerging view from our preliminary studies supports a new model wherein expression of Hox genes from all four complexes participates in limb patterning events, a significant departure from the currently accepted paradigm. We hypothesize that Hox5 genes direct early forelimb AP patterning events by regulating the Tbx5 pathway, and that Hox9 genes are an early signaling node, critical for the establishment of the Shh/Hox10-13 regulatory loop. PUBLIC HEALTH RELEVANCE: Our new genetic results uncover two unexpected and important roles for Hox genes in early limb patterning: Hox5 genes are important for the establishment of anterior limb pattern, and preliminary results suggest they are regulators in the pathway disrupted in human Holt-Oram Syndrome. Further, Hox9 genes appear to be a node for the establishing the Shh-Abdb Hox loop;loss of these genes results in severe truncation of limb development. Analyses of these unexpected phenotypes and functions are critical for developing an understanding of early limb developmental processes and how mis-regulation results in human disease.